N-NITROSAMINES IN RUBBER AND ELASTOMER MATERIALS GB/T 24153

1. Purpose:
1.1 This standard specifies a method for the isolation, identification and determination of N-nitrosamines substances
released from rubber or elastomer and their products.
1.2 N-Nitrosamines are extracted by methanol in ultrasonic bath and analysed for N-nitrosamines by Liquid
Chromatography Mass Spectrometry (LC-MS)

2. Scope:
2.1 To provide detailed analytical methods for the identification and determination of N-nitrosamines released from
rubber and elastomer compiled to the standard method GB/T 24153-2009& EN71-12
2.2 This test method is applicable to rubber or elastomer and their products.

3. Definitions:
3.1 N/A
4. Apparatus and Equipment:
4.1 Gas Chromatography with Mass spectrometer detector (GC-MSD), Agilent HP7890 & MS5975 or equivalent series
4.2 DB-624, 30m x 0.32mm x 1.8µm, J&W or equivalent performance
4.3 Electronic balance, 1 Div. = 0.0001g
4.4 Ultrasonic bath
4.5 Vacuum rotary evaporator
4.6 Cartridges, C-18 cartridges 500mg/ 3mL
4.7 SPE vacuum filtration set up
4.8 Vortex mixer
4.9 100 mL conical flask with ground glass joint fit, amber colour
4.10 150 mL round bottom flask, amber colour
4.11 10 mL centrifuge tube, amber colour
4.12 General laboratory apparatus

5. Reagents, and Consumables:

All chemicals and reagents shall be in analytical grade unless otherwise specified.
5.1 Standards (Chemservice or equivalent quality as follows)
5.1.1 N-nitrosodimethylamine (NDMA), CAS no. 62-75-9 (Accustandard)
5.1.2 N-nitrosomethylethylamine, (NMEA) CAS no. 10595-95-6
5.1.3 N-nitrosodiethylamine (NDEA), CAS no. 55-18-5
5.1.4 N-nitrosodipropylamine (NDPA), CAS no. 621-64-7 (Accustandard)
5.1.5 N-nitrosodibutylamine (NDBA), CAS no. 924-16-3
5.1.6 N-nitrosopiperidine (NPIP), CAS no. 100-75-4
5.1.7 N-nitrosopyrrolidine (NPYR), CAS no. 930-55-2
5.1.8 N-nitrosomorpholine (NMOR), CAS no. 59-89-2
5.1.9 N-nitrosodibenzylamine (NDBzA), CAS no. 5336-53-8 (Accustandard)
5.1.10 N-nitroso N-methyl N-phenylamine (NMPhA), CAS no. 614-00-6
5.1.11 N-nitroso N-ethyl N-phenylamine (NEPhA), CAS no. 612-64-6 (TRC)
5.1.12 N-nitrosodiphenylamine,(NDPhA) CAS no. 86-30-6

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 5.2 Reagents and Solvents
5.2.1 Deionized water, according to ISO 3696:1987, Grade 3
5.2.2 Methanol (HPLC grade)

5.3 Preparation of standard solutions

Note: ALL standards should be protected against light by wrapping in aluminium foil and stored in the dark at a
temperature below 4oC

5.3.1 1000 mg/L individual standard stock solution (storage up to maximum 3 months)
5.3.1.1 Weigh to the nearest 1 mg, 0.01 g of each standard in a 10 mL beaker respectively.
5.3.1.2 Dissolve the standard with small amount of methanol
5.3.1.3 Transfer into a 10 mL amber volumetric flask respectively.
5.3.1.4 Rinse the beaker twice with small amount of methanol (5.3.2).
5.3.1.5 Combine the washings into the volumetric flask and make up to the mark with methanol
(5.3.2).
5.4.2 50 mg/L of intermediate mix standard (mix selected standard)
5.4.2.1 Pipette 0.5mL of each 1000 mg/L individual standard stock solution (5.4.1) into a 10 mL amber
volumetric flask.
5.4.2.2 Make up to the mark with methanol.
5.4.3 5 mg/L of mixed standard spike solution
5.4.3.1 Pipette 1 mL of 10 mg/L mixed intermediate standard solution (5.4.2) into a 10 mL amber
volumetric flask.
5.4.3.2 Make up to the mark with methanol.

5.4 Calibration standard solutions (Freshly prepared daily)

5.4.1 According to Table 1, pipette a volume of 50 mg/L mixed intermediate standard solution (5.4.2) into three
10 mL amber volumetric flask respectively.
5.4.2 Pipette 1mL of 1 mg/L of internal standard working solution (5.5.2) in each flask.
5.4.3 Make up to the mark with methanol.
Table 1. Calibration standard solutions
Volume of 50 mg/L of mixed Final conc. of Final conc. of mixed
intermediate standard solution (mL) Internal standard solution standard solution
(mg/L) (mg/L)

1 0.02 0.1 0.1

2 0.1 0.1 0.5

3 0.2 0.1 1

6. Quality Control:
6.1. A minimum 3 point calibration is used for routine analysis. The correlation coefficient of the calibration curve (R)
shall be greater than 0.995.
6.2 The precision of the instrument is monitored by checking the deviation of the concentration of calibration check
solution. It shall be performed after the calibration and every 20 samples. The acceptance value shall be ± 10% from
the stated value.
6.3 The method blank shall be performed for every batch or every 20 samples, in order to check the contamination. The
acceptance value shall be smaller than the method detection limit.
 6.4 Spike blank / spike sample shall be performed for every batch or every 20 samples. The acceptance value shall be ±
15% from the spike value.
6.5 Duplicates shall be performed for every batch or every 5-20 samples. The acceptance value of the deviation of the
duplicate samples shall be ± 15%.

7. Interferences:
7.1 N-Nitrosamines are degraded by ultra-violet light. Exposure of extracts or standards to sources such as sunlight or
fluorescent tube light should be avoided. Standard or sample should be stored in amber vial, overwrapped in
aluminium foil and stored in dark below 4ºC.
8. Safety and PPE Information:
8.1 Good laboratory practice shall be followed. Use of fume hood, safety glasses, laboratory coat and gloves are
necessary.
8.2 Chemical waste should be disposed into a suitable chemical waste container.
9. Method*:
9.1 Sample preparation and extraction for Nitrosamines
9.1.1 Cut the representative sample into small pieces of about 3mm x 3mm and mix well.
9.1.2 Weigh 1.0 ± 0.01g sample into an amber conical flask (4.9).
9.1.3 Transfer 30 mL of methanol (5.3.2) to the Reaction vial
9.1.4 Close by stopper and put in ultrasonic bath at ambient temperature (25 ± 5)°C for 30 min (± 2 mi n).
9.1.5 Vortex for 1 min to dissolve any residue on containers wall into the extract solution
9.1.6 Close by stopper and put in ultrasonic bath at ambient temperature (25 ± 5)°C for 30 min (± 2 mi n)
9.1.7 Collect the solution filter through PTFE syringe filter to 2 ml reaction vial, injection vial for LC-MS analysis
without delay. If the analysis cannot be performed within 24 hours, the specimen should be kept below –
18oC.
9.1.8 If the concentration of detected nitrosamine in the sample is higher than 10 mg/kg, dilute sample again with
methanol
9.2 Clean up of the extract (in the case of low polluted samples the clean-up step may be omitted)
9.2.1 Set up the SPE filtration and condition the clean up column (4.6)
9.2.2 Rinse with with 5mL methanol (5.3.2).
9.2.3 Elute and discard the washing.
9.2.4 Accurately pipette 2.0 mL sample extract (9.1.12) to the column.
9.2.5 Collect the eluent in a centrifuge tube with graduated mark, stop when the extract has reached the SPE
surface.
9.2.6 Add about 2 mL of methanol onto the column.
9.2.7 Collect all the eluent to the centrifuge tube.
9.2.8 Pre-concentrate the eluant by mean of weak flow inert gas to 2 mL.
9.2.9 Transfer the into a GC vial for LC-MS analysis.

9.3 Preparation of method blank

9.3.1 Without sample involved, perform 9.1.3 to 9.1.17.

9.4 Preparation of spike blank

9.4.1 Spike 0.5 mL of 5 mg/L of mixed standard spike solution (5.4.3) into 30 mL methanol, process 9.1.4 to
9.1.8. The theoretically spike concentration is 0.5 mg/kg (0.5 mg/L)

9.5 Instrumental analysis

 9.5.1 LC-MSD setting (Below instrument conditions are reference only and should be used as a guide)
* Start time of each SIM group is just for reference and should be align after cutting column
9.5.2 Characteristics for LC-MSD analysis

Buffers 0.01% formic acid in

PUMP A
water
0.01% formic acid in
PUMP B
methanol

LC parameters FLOW RATE 0.3

INJECTION VOLUME 20 µL
COLUMN OVEN TEMP. 35°C
Shimpack GISS
COULMN
3.0X50mmX1.7µm

HPLC PROGRAMING:-
TIME MODULE COMMOND VALUE
0.01 Pumps Pump B Conc. 50
5 Pumps Pump B Conc. 50
6 Pumps Pump B Conc. 90
8 Pumps Pump B Conc. 90
9 Pumps Pump B Conc. 50
10 Pumps Pump B Conc. 50
12.01 Controller Stop

Mass PARAMETER:- INTERFACE APCI MODE

NEBULIZING GAS FLOW 3
DRYING GAS FLOW 12
INTERFACE TEMP. 160
DL TEMP. 180
HEAT BLOCK TEMP. 300

Detector Parameter:-
S.NO ANALYTE TARGET ION REFFERENCE ION RT POLARITY
1 NDMA 74.50>58.00 6:MRM(+)
2 NMEA
3 NDEA 103.10>29.15 103.10>27.10-103.10>75.20 0.001 7:MRM(+)
4 NDPA 131.10>43.10 131.10>89.10-131.10>41.20 4.227 3:MRM(+)
5 NDBA
6 NPIP
7 N-Pyrolidine 101.10>55.10 101.10>41.10-101.10>29.10 5:MRM(+)
8 NMOR 117.10>45.05 117.10>87.10-117.10>86.15 2.877 4:MRM(+)
9 NDBzA
10 NMpHA 137.10>66.15 137.10>107.10-137.10>77.10 6.578 1:MRM(+)
11 NMEhA 151.10>77.10 151.10>51.15-151.10>65.95 5.175 2:MRM(+)
12 NDPhA
 9.6 Calibration
9.6.1 Inject 1 µL of each of a series of working standards (5.6) in GC-MSD. Determine the area ratio of each
standard with internal standard. Plot a calibration curve of area response of each standard against its
concentration.
9.7 Sample measurement
9.7.1 Inject 1 µL of the sample solution, method blank and spike blank in GC-MSD and obtain area ratio in each
analytes with internal standard. From the calibration curve, read off the concentration of each analytes in
sample solution corresponding to the area ratio. 9.8 Calculation

CxV
Each analyte in sample, mg/kg =
D.F. x W
where
C = Concentration of analyte in sample extract, mg/L
W = Weigh of sample, 5 g
V = Final volume, 5 mL
D.F. = Dilution factor, if applicable

10. Reporting Criteria*:

10.1 Reporting limit is shown at the following table

Substances Laboratory Reporting Limit (mg/kg)

N-nitrosodimethylamine (NDMA) 0.5

N-nitroso methyl-ethylamine (NMEA) 0.5

N-nitrosodiethylamine (NDEA) 0.5

N-nitroso N-methyl N-phenylamine (NMPhA) 0.5

N-nitrosodipropylamine (NDPA) 0.5

N-nitrosomorpholine (NMOR) 0.5

N-nitrosopyrrolidine (NPYR) 0.5

N-nitroso N-ethyl N-phenylamine (NEPhA) 0.5

N-nitrosopiperidine (NPIP) 0.5

N-nitroso-diphenylamine (NDPhA) 0.5

N-nitrosodibutylamine (NDBA) 0.5

N-nitrosodibenzylamine (NDBzA) 0.5

10.2 The MDL for each N-Nitrosamine is 0.1 mg/kg.

11. Measurement Uncertainty:

11.1 Measurement uncertainty needs to be calculated by location themselves if necessary
12. Literature References:
12.1 GB/T 24153-2009 Rubber and elastomer materials—Determination of N-nitrosamines
13. Modifications from Standard:
13.1 Adopted use of internal standard.
 13.2 Non-compulsory use of clean up column.
14. Associated Documents:
15. Annex:

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